Recent human studies report an emerging relationship between the gut microbiota and its metabolites with the development of atherosclerotic disease and adverse cardiovascular (CV) events. However, critical knowledge gaps must be bridged to translate these foundational works into specific, gut microbiota-targeted interventions to reduce CV risk. These include 1) identifying mechanisms by which a selected intervention impacts the vascular endothelial function and mediators of atheroma formation and 2) identifying differences in the impact of a selected intervention on key populations with known differences in gut flora (including sex and obesity status differences). This proposal will address these issues for L. plantarum 299v (Lp299v) supplementation. Our preliminary data in 20 men with coronary artery disease (CAD) suggest that probiotic supplementation with Lp299v has a strong and favorable impact on the vascular endothelium and a strong anti-inflammatory effect on inflammatory cells critical to the development of endothelial dysfunction and atherosclerosis. Our preliminary data suggest obese CAD patients derive the greatest benefit, but whether the favorable impact of Lp299v supplementation systematically differs in women or in obese humans with CAD remains unknown and will be the foci of Aims 1 and 2. Our preliminary data also show improved endothelium dependent vasodilation and reduced systemic inflammation occur concomitantly with changes in the concentrations of circulating short-chain fatty acid (SCFAs)? known systemic metabolic products of the gut microbiota. In animal models, SCFAs cause endothelium-dependent vasodilation by activating G protein-coupled receptor 41 (FFAR3). Whether this novel mechanism is relevant in humans with CAD remains unknown. Our preliminary data shows Lp299v supplementation increases circulating propionate, a three-carbon SCFA. Additionally, post- supplementation plasma reversed impaired endothelium-dependent vasodilation in resistance arteries from CAD patients in an eNOS- and FFAR3-dependent manner. FFAR3's role in endothelium-dependent vasodilation responses to Lp299v is a focus of in Aim 2. In Aim 3, we will test whether Lp299v supplementation reduces pro-inflammatory signaling in human mononuclear cells known to contribute to atherosclerosis formation and disease activity and whether FFAR3 activity is involved in this effect. The application employs an innovative mix of translational investigations. We combine a randomized clinical trial to study sex- and BMI-specific effects of Lp299v on vascular function using brachial artery ultrasound with critical molecular and pharmacological studies targeting FFAR3 expression and activity in intact human vessels and mononuclear cells. Additionally, we will employ innovative plasma-induced transcription studies and pathway analyses to determine the impact of supplementation on whole genome transcription in human mononuclear cells [lymphocytes (T-cell, B-cell, and NK cells) and monocytes] to determine how Lp299v impacts key regulatory pathways involved in human vascular inflammation, atheroma formation, and plaque stability.